Nicotine is a potent stimulator of the secretion of two stress- responsive hormones, ACTH and prolactin. Studying its pharmacological effects on these two neuroendocrine axes has also shown that nicotine inhibits some centrally-mediated stress responses in rats. Recent experiments suggest that these effects of nicotine may reflect its direct action on the CNS. The proposed investigations are designed to elucidate the neurochemistry that mediates these events by focusing on key elements of the neuronal systems that mediate the stimulative effects of nicotine on ACTH and prolactin secretion. Studies are designed to locate the brain nuclei where nicotine is active, using chronic intracerebral nuclear (ICN) cannulation of hypothalamic and brainstem sites. Serotonin is hypothesized to be a critical neurotransmitter released by nicotine which regulates the level of hypothalamo- hypophysial oxytocin controlling ACTH and prolactin secretion. Therefore, studies with selective antagonists of oxytocin and serotonin, and selective serotonergic neuronal ablation by 5,7 DHT are proposed. Mediation of nicotine action by catecholaminergic afferent inputs to the hypothalamus will also be evaluated using antagonists administered both ICN and peripherally, and neuronal ablation by 6, OHDA. Two classes of central nicotinic cholinergic binding sites, labeled by 125I-alpha-bungarotoxin (BTX) versus 3H- nicotine, have been reported; these are present in key nuclear regions within the hypothalamus. K-BTX which appears to inhibit the depolarization of ganglia mediated by 3H-nicotine-like binding sites, will be purified to help 3 determine the functional significance of central nicotinic binding sites. Displacement of 3H-nicotine binding to brain homogenates by K-BTX will be assessed. Then, K and alpha-BTX will be instilled ICN to identify the receptor(s) which are linked to nicotine-induced hormonal secretion. Monoclonal antibodies (Mab), to a purified high affinity binding site for 3H-nicotine/3H-acetycholine and to oligopeptides derived from a C-DNA coding for a similar binding site, are also being generated to identify the functional binding site(s). Mab binding to rat brain homogenates will undergo extensive characterization. Moreover, a radioimmunohistochemical map of Mab binding to brain sections will be compared quantitatively to the map of 125I-alpha-bungarotoxin versus 3H- nicotine binding sites. Insights into the mechanism of nicotine action in the CNS gained from these experiments should provide a firm basis for future investigation of the mechanism(s) underlying the stress-attenuative effects of nicotine.